DNA or RNA encoding a recombinant dengue envelope protein

ABSTRACT

A recombinant protein encompassing the complete envelope glycoprotein and a portion of the carboxy-terminus of the membrane/premembrane protein of dengue 2 virus was expressed in baculovirus as a protein particle. The recombinant protein particle was purified and found to provide protection against lethal challenge with dengue 2 virus in mice.

BACKGROUND OF THE INVENTION

This is a divisional application of Ser. No. 09/468,517 filed Dec. 21,1999, now U.S. Pat. No. 6,514,501, which is a divisional of Ser. No.08/504,878, filed Jul. 20, 1995, now U.S. Pat. No. 6,074,865.

GOVERNMENT INTEREST

The invention described herein may be manufactured, used and licensed byor for the U.S. Government.

INTRODUCTION

This invention relates to the production and purification of arecombinant protein for use as a diagnostic tool and as a vaccineagainst Dengue virus.

Dengue (DEN) viruses are human pathogens with a significant threat toworld health. These viruses are estimated to cause several hundredthousand cases of dengue fever, dengue hemorrhagic fever (DHf) anddengue shock syndrome (DSS) annually (Shope, R. E. In: The Togaviruses.Schlesinger, R. W. (Ed.) Academic Press, New York. 1980, pp. 47-82;Monath, T. P. In: The Togaviridae and Flaviviridae, Schlesinger, S. andSchlesinger, M. J. (Eds.) New York and London, 1986, pp. 375-440;Halstead, S. B. Bull. W.H.O. 1980, 58, 1-21; Halstead, S. B. Am. J.Epidemiol. 1984, 114, 632-648) The complete content of all documentscited herein are hereby incorporated by reference. Dengue viruses aremembers of the family Flaviridae and are transmitted by Aedes mosquitoes(Halstead, S. B. Science 1988, 239, 476-481). There are four serologicaltypes, DEN-1, DEN-2, DEN-3 and DEN-4, distinguishable bycomplement-fixation assays (Sabin, A. B. and Young, I. A. Proc. Soci.Exp. Biol. Med. 1949, 69, 291-296), virus plaque-reductionneutralization tests (Russell, P. K. and Nisalak, A. J. Immunol. 1967,99, 291-296) and immunoassays using monoclonal antibodies (MAbs)(Gentry, M. K. et al. Am. J. Trop. Med. Hyg. 1982, 31, 548-555; Henchal,E. A. et al. Am. J. Trop. Med. Hyg. 1982, 31, 830-836).

Dengue viruses are composed of a single-stranded RNA molecule ofpositive polarity (messenger sense) which is contained within anucleocapsid composed of capsid (C) protein. The capsid is surrounded bya lipid envelope about 50 nm in diameter in which are embedded theenvelope (E) glycoprotein and the matrix (M) protein. Both thestructural and nonstructural (NS) proteins are encoded by a single, longopen reading frame of about 10.5 kilobases arranged as follows:C-PreM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS5 (Rice, C. M. et al. Science 1985,229, 726-733; Wengler, G. et al. Virology 1985, 147, 264-274; Castle, E.et al. Virology 1986, 149, 10-26; Zhao, B. et al. Virology 1986, 155,77-88; Mason, P. W. et al. Virology 1987, 161, 262-267; Mackow, E. etal. Virology 1987, 159, 217-228; Sumiyoshi, H. et al. Virology 1987,161, 497-510; Irie, K. et al. Gene 1989, 74, 197-211).

Attempts to prevent DEN virus infection have focused on the productionof a vaccine which would protect against all four serotypes. However,despite more than 50 years of effort, safe and effective dengue virusvaccines have not been developed. Candidate vaccines currently beingtested fall into two categories: live attenuated dengue virus vaccinesand subunit vaccines, each with its own drawbacks.

Live attenuated virus vaccines have been demonstrated to be eitherunder-attenuated (cause disease) or over-attenuated (fail to immunize).Even an optimally-attenuated live virus vaccine can revert to a virulent(disease-causing) form through mutation. Live dengue viruses are alsosensitive to heat, making it difficult and costly to maintain thevaccine in some tropical and subtropical countries where the vaccine maybe needed most.

Recombinant subunit vaccines have the advantage of eliminating the riskof infectivity and greater chemical stability. However, the subunitvaccines of flavivirus structural and NS proteins produced in expressionvectors including baculovirus, vaccinia virus and E. coli reported sofar elicit only low titers of neutralizing antibody and are difficult toproduce in large quantities and pure form (Putnak, J. R. et al. Virology1988, 163, 93-103; Putnak, J. R. et al. Am. J. Trop. Med. Hyg. 1991, 45,159-167; Zhang, Y. M. et al. J. Virol. 1988, 62, 3027-3031; Lai, C. J.et al. In: Vaccines, Modern Approaches to New Vaccines IncludingPrevention of AIDS (Eds. Lerner, R. A. et al.), Cold Spring HarborLaboratory Press, New York, 89, 1989, pp. 351-356; Bray, M. et al. J.Virol. 1989, 63, 2853-2856; Bray, M. and Lai, C. J. Virology 1991, 185,505-508; Men, R et al. J. Virol 1991, 65, 1400-1407; Mason, P. W. et al.Virology 1987, 158, 361-372; Mason, P. W. et al. J. Gen. Virol. 1989,70, 2037-2049; Mason, P. W. et al. J. Gen. Virol. 1990, 71, 2107-2114;Murray, J. M. et al. J. Gen. Virol., 1993, 74, 175-182; Preugschat, F.et al. J. Virol. 1990, 64, 4364-4374).

Both the envelope (E) and the nonstructural protein 1 (NS1) arecandidates for recombinant, subunit vaccines against DEN virus. The Eglycoprotein is the major surface protein of the virion. It functions invirion attachment to host cells and it can be detected by its ability tohemagglutinate goose erythrocytes. As an antigen, it containsvirus-neutralizing epitopes (Stevens, T. M. et al. Virology 1965, 27,103-112; Smith, T. J. et al. J. Virol. 1970, 5, 524-532; Rice, C. M. andStrauss, J. H. J. Mol. Biol. 1982, 154, 325-348; Brinton, M. A. In:Togaviridae and Flaviridae. Schlesinger, S. and M. J. Schlesinger(Eds.), M. J. Plenum, New York, 1986, pp. 327-365; Heinz, F. X. Adv.Virus Res. 1986, 31, 103-168; Westaway, E. G. Adv. Virus Res. 1987, 33,45-90; Hahn, Y. S. et al. Arch. Virol. 1990, 115, 251-265). Neutralizingantibodies, believed to correlate with protection, andhemagglutination-inhibiting (HI) antibodies develop following naturalinfection. Mice immunized with purified DEN-2 E antigen developneutralizing antibodies and are protected against lethal virus challenge(Feighny, R. J. et al. Am. J. Trop. Med. Hyg. 1992, 47, 405-412).

Recombinant DEN proteins have been produced using the baculovirus systemfor the purpose of developing a vaccine. Results have been variable andsometimes disappointing. Several stategies have been used to produce theDEN E protein in the baculovirus system. One strategy used a truncatedgene to produce the E protein without the hydrophobic transmembranesegment of the carboxy terminus. The purpose of this approach was topromote secretion and solubility of the protein. Proteins produced inthis manner were minimally immunogenic in mice (Putnak, R. et al. Am. J.Trop. Med. Hyg., 1993, 45: 159-167; Zhang, Y. M. et al., J. Virol.,1988, 62: 3027-3031). Another strategy used a polygene that encoded thecapsid, premembrane and two nonstructural proteins, C-prM-E-NS1-NS2(Delenda et al. J. Gen. Virol., 1994, 75: 1569-1578). This constructproduced the full length E protein by cleavage of the polyprotein.Neutralizing antibody to the full length E protein was not elicited bythat product although protection was induced. The complex nature of theconstruct precludes an analysis of the reason for protection in theabsence of neutralizing antibody but the presence of NS1 in theconstruct was speculated to have induced the protective response.Another strategy employed a construct that contained a polygene encodingC, preM and a truncated E protein (Deubel et al. Virology, 1991, 180:442-447). Although the truncated E reacted with some E-specificmonoclonal antibodies (mAbs), reactivity was weaker than that obtainedwith native virus.

Therefore, in view of the problems with the presently available vaccinesdiscussed above, there is a need for a DEN vaccine that elicits veryhigh titers of neutralizing antibody, provides protection against thedisease, has no possibility of infectivity to the immunized host, can beproduced easily in pure form, and is chemically stable.

SUMMARY

The present invention is directed to a subunit vaccine that satisfiesthis need. The recombinant DEN virus subunit vaccine of the presentinvention comprises the full dengue virus envelope protein, expressed inbaculovirus and capable of self-assembling into a particle. Dengueenvelope protein has been expressed in the baculovirus system by others.The previously produced products were poorly immunogenic when tested inanimals. None of the previously made products are known to formparticles. The protein is expressed and purified as a particle composedof multiple dengue envelope protein molecules. Particles are moreimmunogenic than soluble proteins, possibly because they can crosslinkcell surface immunoglobulins on B cells. The envelope protein particleof the present invention is produced in baculovirus in large quantitiesand in pure form, elicits high titers of neutralizing antibody and isprotective against the disease in the immunized animal.

The present invention describes the production of the DEN envelopeprotein particle by cloning the complementary DNA (cDNA) sequencesencoding the envelope protein fragment into an expression vector suchthat the recombinant dengue protein can be expressed. The recombinantprotein is produced in baculovirus, isolated and purified as a particlewhich is antigenic, reactive with dengue virus-specific and monoclonalantibodies and capable of eliciting the production of neutralizingantibodies when inoculated into mice. The administration of thisrecombinant subunit vaccine is demonstrated to protect mice, an acceptedanimal model, against morbidity and mortality following challenge withlive dengue virus.

Therefore, it is an object of the present invention to provide a DEN 2cDNA fragment encoding the full envelope glycoprotein, said genecontaining 1485 nucleotides plus 93 adjacent upstream sequences andextending from 844 to 2422 of the viral genome and is useful as adiagnostic agent and a naked DNA vaccine.

It is another object of the invention to provide a recombinant vectordesigned to produce the recombinant DEN envelope protein for use as avaccine and as a diagnostic agent.

It is still another object of the invention to provide a purified DENenvelope protein particle useful as a vaccine against DEN disease andfor detecting the presence of said disease in a suspected patient.

It is another object of the present invention to provide a method forthe purification of recombinant DEN envelope protein particle for use asa vaccine or as a diagnostic tool.

It is yet another object of the invention to provide a DEN virus vaccineeffective for the production of antigenic and immunogenic responseresulting in the protection of an animal against dengue virus disease.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

FIG. 1. Illustration of the pBlueBacIII shuttle vector and genesequences used for expression of the dengue 2 virus envelopegylcoprotein in insect cells. A) illustration of relative positions ofdengue 2 virus structural protein genes capsid©, premembrane (prM) andenvelope (E), and the N-terminal end of the adjacent non-structuralprotein NS1; B) nucleotide coordinates of the E gene construct used forinsertion into shuttle vector pBluBacIII, extending from nucleotides 844to 2422, including a sequence from vector identifying relative positionsof the beta-galactosidase gene (lacZ), polydedrin promoter (Pph),BglII/PstI cloning site, recombination sequences and ampicillinresistance gene (amp).

FIG. 2. Gel filtration of dengue 2 virus recombinant envelopeglycoprotein (rEgp) expressed by baculovirus using a column of G100Sephadex. The column was equilibrated in phosphate buffered saline (PBS)and fractions were eluted in PBS. Fractions were assayed for antigenicreactivity using the antigen dot blot assay and hyperimmune murineascites fluid specific for dengue 2 virus. Data are plotted asabsorbance at 280 nanometers (A260 nm) and counts per minute yesesfraction number. Solid line represents relative absorbance of the sampleat 280 nm; dashed line represents antigenic activity; dotted linerepresents the elution pattern of column calibration standardsthyroglobulin, 670 kilodaltons (kD), bovine gamma globulin, 158 kD,chicken ovalbumin, 44 kD and myoglobin, 17 kD.

FIG. 3. Chromatographic analysis of recombinant dengue 2 virus envelopeglycoprotein (rEgp) expressed by baculovirus using fast pressure liquidchromatography (FPLC) and a Superose 6 column. The column wasequilibrated with phosphate buffered saline (PBS) and protein was elutedwith the same. A. Column fractions were assayed for antigen usinganti-dengue 2 hyperimmune ascited fluid in a dot blot assay. Data areplotted as absorbance at 280 nanometers (A260 nm) and counts per minute(y axis) vesus fraction number. B. The column as calibrated withmolecular weights standards thyroglobulin, 670 kilodaltons (kD), bovinegamma globulin, 158 kD, chicken ovalbumin, 44 kD and myoglobin, 17 kD.

FIG. 4. Effect of sarkosyl on chromatographic elution profile ofrecombinant dengue 2 virus envelope glycoprotein (rEgp) analyzed using aSuperose 6 column and fast pressure liquid chromatography (FPLC). Thecolumn was equilibrated in phosphate buffered saline (PBS) containing0.1% sodium sarkosyl and protein containing recombinant dengue 2envelope glycoprotein was eluted in PBS containing: A) 0.1% sarkosyl, B)1.0% sarkosyl, C) 2.0% sarkosyl and D) 3.0% sarkosyl. Column fractionswere assayed for antigenic activity using anti-dengue 2 hyperimmuneascites fluid in a dot blot assay. Data are plotted as absorbance (solidline) at 280 nanometers (A260) and counts per minute (dotted line) vesusfraction.

FIG. 5. Effect of sonication on chromatographic elution profile ofrecombinant dengue 2 virus envelope glycoprotein (rEgp) analyzed usisnga Superose 6 column and fast pressure liquid chromatography (FPLC).Insect cells (Trichoplusia ni) infected with recombinant baculovirusexpressing the dengue 2 virus envelope glycoprotein were sonicated inphosphate buffered saline (PBS) for 0, 20 and 30 minutes and eluted froma Superose 6 column by fast pressure liquid chromatography (FPLC). Solidline represents relative amounts of protein detected by absorbancy at280 nanometers (A260) and dotted line (counts per minute) representsantigenic reactivity of fraction aliquots with anti-dengue 2 hyperimmuneascites fluid in a dot blot assay.

FIG. 6. Sucrose gradient centrifugation distribution of recombinantdengue 2 virus envelope glycoprotein (rEgp). Insect cells (Spodopterafrugiperda) infected with recombinant baculovirus were pelleted at lowspeed and protein remaining in the supernatant was pelleted at 100,000×g for 2.5 hours. The resulting microsomal pellet was subjected todensity gradient ultracentrifugation at 100,000 ×g for 2.5 hours using astp gradient of 5-30% sucrose in phosphate buffered saline (PBS).Fractions were assayed for antigenic activity (shaded area) usinganti-dengue 2 hyperimmune ascites fluid in a dot blot assay.

FIG. 7. Polyacrylamide gelelectrophoresis and immunoblot analysis ofbaculovirus-expressed dengue 2 virus recombinant envelope glycoprotein(rEgp). The microsomal pellet (described, FIG. 6) was ultracentrifugedthrough a cushion of 30% sucrose in phosphate buffered saline (PBS) for2.5 hours at 100,000 ×g. Proteins in the microsomal pellet or 30%sucrose pellet were resuspended in PBS, sonicated briefly and boiled inSDS sample buffer for 5 minutes before electrophoresis on a 10% SDSpolyacrylamide gel. A) Coomassie-blue stained gel: lane 1, molecularweight standard; lane 2, microsomal pellet; lanes 3 and 4, 30% sucrosepellets (contained in 10 or 20 microliters respectively). B) Proteinswere electrophoretically transferred to nitrocellulose paper and thisimmunoblot was probed with hyperimmune mouse ascites fluid specific fordengue 2 virus. Lanes in B correspond to lanes in A.

DETAILED DESCRIPTION

In one embodiment, the present invention relates to a DNA or cDNAsegment which encodes the complete E protein of DEN-2 and the carboxyterminus of membrane/premembrane protein extending from nucleotide 844to 2422 of the DEN-2 viral genome and including linear andconformational, neutralizing epitopes said sequence identified as SEQ IDNO: 1.

DNA sequences to which the invention also relates include sequenceswhich encode the specific protein epitopes within said sequence whichelicit neutralizing antibody production in animals upon administrationof the protein encoded by said DNA sequences. Specifically, suchsequences include regions encoding neutralizing epitopes present on thenucleotide sequence encompassing amino acids 1 through 495 of the Eprotein several of which have been mapped (Henchel, E. et al. Am. J.Trop. Med. Hyg., 1985, 34:162-167) and found to be conformational aswell as linear epitopes examples of which are found in TABLE 1 underResults section.

In another embodiment, the present invention relates to a recombinantDNA molecule that includes a vector and a DNA sequence as describedabove (advantageously, a DNA sequence encoding the protein having theneutralizing antibody-eliciting characteristics of that protein). Thevector can take the form of a virus shuttle vector such as, for example,baculovirus vectors pBlueBac-III, pBlueBac-HIS-A-B-C, MaxBac; a plasmid,or eukaryotic expression vectors such as such as GST gene fusionvectors, pGEx-3x, pGEx-2T, pGEx, mammalian cell vectors (pMSG, pMAMneo)or vectors for expression in drosophila or yeast, in addition to othervectors known to people in the art. The DNA sequence can be present inthe vector operably linked to regulatory elements, including, forexample, a promoter or a highly purified human IgG molecule, for exampleProtein A, an adjuvant, a carrier, or an agent for aid in purificationof the antigen as long as the rEgp is expressed as a particle. Therecombinant molecule can be suitable for transforming transfectingeukaryotic cells for example, mammalian cells such as VERO or BHK cells,or insect cells such as Sf-9 (Spodopter frugiperda), C6/36 (Aedesalbopictus), and Trichoplusia ni (High five) mosquito cells, Drosophilacells, and yeast (Ssccharomyces cerevisiae) among others.

In another embodiment, the present invention relates to a recombinantprotein having an amino acid sequence corresponding to SEQ ID NO: 2 andencompassing 495 amino acids of the E protein and 36 amino acids of thecarboxy-terminus of the adjacent M/prM protein from DEN-2 or any allelicvariation thereof which maintains the neutralizing antibody productioncharacteristic of the recombinant protein. As an example, the protein(or polypeptide) can have an amino acid sequence corresponding to anepitope such as a B-cell and T-cell epitope present on the envelopeglycoprotein of DEN-2, or conformational epitopes examples of which arefound in TABLE 1. In addition, the protein or polypeptide, or a portionthereof, can be fused to other proteins or polypeptides which increaseits antigenicity, thereby producing higher titers of neutralizingantibody when used as a vaccine. Examples of such proteins orpolypeptides include any adjuvants or carriers safe for human use, suchas aluminum hydroxide and liposomes.

In yet another embodiment, the present invention relates to arecombinant protein as decribed above which is capable of assemblinginto more than one protein unit. Assembly of the individual proteinunits can be by hydrophobic forces, or chemical forces, by cross-linkingreagents, or the assembled protein can be further stabilized bycross-linking reagents, and liposomes. The particle can encompass fromat least 2 units of envelope protein. Such a particle can provide higherimmunogenicity and possibly cross-link cell surface immunoglobulins on Bcells.

In a further embodiment, the present invention relates to host cellsstably transformed or transfected with the above-described recombinantDNA constructs. The host cell can be lower eukaryotic (for example,yeast or insect) or higher eukaryotic (for example, all mammals,including but not limited to mouse and human). For instance, transientor stable transfections can be accomplished into CHO or Vero cells.Transformation or transfection can be accomplished using protocols andmaterials well known in the art. The transformed or transfected hostcells can be used as a source of the DNA sequences described above. Whenthe recombinant molecule takes the form of an expression system, thetransformed or transfected cells can be used as a source of theabove-described recombinant protein.

In a further embodiment, the present invention relates to a method ofproducing the recombinant protein which includes culturing theabove-described host cells, under conditions such that the DNA fragmentis expressed and the recombinant protein is produced thereby. Therecombinant protein can then be isolated using methodology well known inthe art. The recombinant protein can be used as a vaccine for immunityagainst infection with flaviviruses or as a diagnostic tool fordetection of viral infection.

In yet another embodiment, the present invention relates to a method ofpurifying the recombinant protein particles, said method comprising thesteps of:

(i) harvesting cells expressing recombinant DEN envelope glycoprotein;

(ii) separating a cell pellet and a supernatant from said harvestedcells;

(iii) lysing said cell pellet of step (ii) to release recombinantenvelope glycoprotein;

(iv) pelleting said recombinant envelope glycoprotein from said lysedcells;

(v) fractionating said recombinant envelope glycoprotein from steps (ii)and (v) through a density gradient;

(vi) collecting purified recombinant envelope glycoprotein from pellet.

The density gradient of step (vi) may be made of any density separationmaterial such as cesium chloride, ficoll, or molecular sieve material.The recombinant envelope glycoprotein can also be pelleted from saidsupernatant. If desired, the cell debris can be pelleted or separatedfrom said recombinant envelope glycoprotein after lysing cell pellet asdescribed in (iii).

In a further embodiment, the present invention relates to a method ofdetecting the presence of DEN virus disease or antibodies against DENvirus in a sample. Using standard methodology well known in the art, adiagnostic assay can be constructed by coating on a surface (i.e. asolid support) for example, a microtitration plate or a membrane (e.g.nitrocellulose membrane), all or a unique portion of the recombinantenvelope protein particle described above, and contacting it with theserum of a person suspected of having DEN fever. The presence of aresulting complex formed between the recombinant protein and antibodiesspecific therefor in the serum can be detected by any of the knownmethods common in the art, such as fluorescent antibody spectroscopy orcolorimetry. This method of detection can be used, for example, for thediagnosis of DEN disease. This method when employing distinct rEgpparticles specific for each DEN serotype, will allow the detection ofthe presence of each respective DEN serotype in a sample. Infection withmore than one serotype is thought to play a role in the etiology of DENhaemorrhagic fever and DEN shock syndrome.

In addition, the present invention is related to a method of detectingflavivirus disease or antibodies against flavivirus in a sample. Dengueviruses are members of the family Flaviridae which includes over sixtymembers among which there is considerable genetic and antigenicsimilarity but no significant cross-neutralization. It would be apparentto persons in the art to apply the concepts of the present inventionexemplified in DEN-2 to similar proteins and DNA sequences present inother related flaviviruses such as yellow fever, Japanese encephalitisand tick-borne encephalitis viruses.

In another embodiment, the present invention relates to a diagnostic kitwhich contains the recombinant envelope protein particle and ancillaryreagents that are well known in the art and that are suitable for use indetecting the presence of antibodies to flavivirus antigens in serum ora tissue sample, specifically antibodies to DEN virus. Tissue samplescontemplated can be monkey and human, or other mammals.

In another embodiment, the present invention relates to a vaccine forprotection against a flavivirus disease. The vaccine can be prepared byinducing expression of the recombinant expression vector described abovein either a higher mammalian or lower (insect, yeast, fungi) eukaryotichost and purifying the recombinant glycoprotein particle describedabove. The purified particles are prepared for administration to mammalsby methods known in the art, which can include preparing the particleunder sterile conditions and adding an adjuvant. The vaccine can belyophilized to produce a flavivirus vaccine in a dried form for ease intransportation and storage. Further, the vaccine may be prepared in theform of a mixed vaccine which contains the recombinant protein describedabove and at least one other antigen as long as the added antigen doesnot interfere with the effectiveness of the dengue vaccine and the sideeffects and adverse reactions are not increased additively orsynergistically. It is envisioned that a tetravalent vaccine composed ofrecombinant antigenic proteins from the four serotypes of dengue virus,DEN-1, DEN-2, DEN-3, and DEN-4 can be produced to provide protectionagainst dengue disease.

The vaccine may be stored in a sealed vial, ampoule or the like. Thepresent vaccine can generally be administered in the form of a liquid orsuspension. In the case where the vaccine is in a dried form, thevaccine is dissolved or suspended in sterilized distilled water beforeadministration. Generally, the vaccine may be administeredsubcutaneously, intradermally or intramuscularly in a dose effective forthe production of neutralizing antibody and protection from infection.

In another embodiment, the present invention relates to a naked DNA orRNA vaccine. The DEN DNA fragment, of the present invention described inSEQ ID NO: 1 or a portion thereof, or an allelic form thereof, can beadministered as a vaccine to protect against DEN virus disease and toelicit neutralizing antibodies against the virus. The DNA can beconverted to RNA for example by subcloning the said DNA into atranscriptional vector, such as pGEM family of plasmid vectors, or undercontrol of a transcriptional promoter of a virus such as vaccinia, andthe RNA used as a naked RNA vaccine. It is understood and apparent to aperson with ordinary skill in the art that due to the similarity betweendifferent serotypes of DEN as well as similarities between flaviviruses,a DNA sequence from any DEN serotype or flavivirus encoding the completeenvelope protein of its respective flavivirus can be used as a naked DNAvaccine against infection with its respective virus. The DEN-2 naked DNAor RNA vaccine can be injected alone, or combined with at least oneother antigen or DNA or RNA fragment as long as the added antigen or DNAor RNA fragment does not interfere with the effectiveness of the DENvaccine and the side effects and adverse reactions are not increasedadditively or synergistically. It is envisioned that a tetravalentvaccine composed of DNA or RNA fragments from the four serotypes ofdengue virus, DEN-1, DEN-2, DEN-3, and DEN-4 can be produced to provideprotection against dengue disease.

The naked DNA or RNA vaccine of the present invention can beadministered for example intermuscularly, or alternatively, can be usedin nose drops. The DNA or RNA fragment or a portion thereof can beinjected as naked DNA or RNA, as DNA or RNA encapsulated in liposomes,as DNA or RNA entrapped in proteoliposomes containing viral envelopereceptor proteins (Nicolau, C. et al. Proc. Natl. Acad. Sci. U.S.A.1983, 80, 1068; Kanoda, Y., et al. Science 1989, 243, 375; Mannino, R.J. et al. Biotechniques 1988, 6, 682). Alternatively, the DNA can beinjected along with a carrier. A carrier can be a protein or such as acytokine, for example interleukin 2, or a polylysine-glycoproteincarrier (Wu, G. Y. and Wu, C. H. J. Biol. Chem. 1988, 263, 14621), or anonreplicating vector, for example expression vectors containing eitherthe Rous sarcoma virus or cytomegalovirus promoters. Such carrierproteins and vectors and methods for using same are known to a person inthe art (See for example, Acsadi, G. et al. Nature 1991, 352, 815-818).In addition, the DNA or RNA could be coated onto tiny gold beads andsaid beads introduced into the skin with, for example, a gene gun(Cohen, J. Science 1993, 259, 1691-1692; Ulmer, J. B. et al. Science1993, 259, 1745-1749).

Described below are examples of the present invention which are providedonly for illustrative purposes, and not to limit the scope of thepresent invention. In light of the present disclosure, numerousembodiment within the scope of the claims will be apparent to those ofordinary skill in the art.

The following MATERIALS AND METHODS were used in the examples thatfollow.

Cells and Viruses.

Dengue-2 virus was propagated in Aedes albopictus cells (C6/36 cells,American Type Tissue Culture Collection, ATCC, Rockville, Md.). Topropagate virus, C6/36 cells were grown at 28° C. in CO₂-independentmedium (Gibco, Grand Island, N.Y.) containing 10% fetal bovine serum(FBS, heat inactivated at 56° C. for 30 min, Sigma, St Louis, Mo.).Wild-type DEN-2 virus (strain PR 159) was the source of genomic RNA forsynthesis of the rEgp gene. A mouse-adapted New Guinea C strain was usedfor immunizations and plaque neutralization assays. African green monkeykidney cells were purchased from ATCC. Baculovirus (Autographacalifornica nuclear polyhedrosis virus, AcPNV, Invitrogen, San Diego,Calif.) was propagated in Spodoptera frugiperda (Sf-9 and Sf-21) andTrichoplusia ni (High five) cells (Invitrogen). High five cells and Sf-9cells were cultured in tissue culture flasks at 28° C. in TNMFH medium(Biowhittaker, Walkersville, Md.) supplemented with 10% FBS, penicillin(100 units U/ml), streptomycin (100 μg/ml), glutamine (2 mM) andgentamycin (50 mg/ml). Recombinant baculoviruses were isolated in Sf-9cells following previously described procedures (5). The Sf-21 cellswere grown in 10 liter spinner culture in TNMFH media supplemented asabove for High five and Sf-9 cells.

Cloning of the DEN-2 Envelope Gene.

The gene encoding the DEN-2 Egp and an adjacent upstream translocationsignal sequence (Markoff, L, J. Virol., 1989, 63:3345-3352.) was derivedby reverse transcription of viral genomic RNA followed by amplificationof cDNA by the polymerase chain reaction. Dengue-2 virus RNA waspurified from supernatants of virus-infected C6/36 cells by guanidineisothiocyanate-phenol chloroform:isoamyl alcohol extraction (Chomczynskiand Sacchi, Anal. Biochem., 1987, 162:156-159). Primers were constructedthat incorporated enzyme restriction sequences onto ends of the Egp genefragment, and the fragment was inserted into the baculovirus transfervector pBlueBacIII (Invitrogen). The sequence of the recombinant Egp(rEgp) gene fragment in pBlueBacIII was determined to be identical tothat of the native Egp gene (Hahn, et al. Virology, 1988, 185:401-410)by dideoxy sequencing (Sanger et al., Proc. Natl. Acad. Sci. U.S.A.,1977, 74: 5463-5467).

Cotransfection and Purification of Recombinant Baculoviruses.

Recombinant baculoviruses were generated by co-transfecting Sf-9 cellswith a recombinant pBlueBac III plasmid together withcommercially-prepared linear baculovirus (Invitrogen, San Diego,Calif.). The Egp gene fragment was transferred into the baculovirusgenome by homologous recombination (Summers and Smith, A Manual ofMethods for Baculovirus Vectors and Insect Cell Culture Procedure. TexasAgricultural Experimental Station Bulletin No. 1555, Texas AgriculturalStation, College Station, Tex., 1987). Plaque assays in Sf-9 cells wereused to isolate the recombinant baculovirus clones which yielded blueplaques due to the transfer of the β-galactosidase gene from thepBlueBac III plasmid. Following infection of Sf-9 cells with a plaquepurified recombinant baculovirus clone, DNA was extracted from cells andthe presence of the Egp gene was confirmed by hybridization of a³²P-labeled Egp gene probe with the DNA.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting.

Proteins were resolved on a 10% SDS-polyacrylamide gel (Laemmli, U.K.Nature, 1970, 227:680-685). Samples were either boiled for 5 minutes ornot boiled before application to the gel. Proteins were blotted ontonitrocellulose paper using a dry blot apparatus (Enprotech, IntegratedSeparation Systems, Hyde Park, Mass.) as recommended by themanufacturer. Following protein transfer, the nitrocellulose was blockedfor 30 minutes in PBS-0.05% azide containing 5% powdered milk (blockingbuffer) and incubated overnight in blocking buffer containing a 1:500dilution of anti-DEN-2 hyperimmune mouse ascites fluid (HMAF, 11). Theblot was washed 3 times in PBS containing 0.05% Tween 20 (PBS-T) andincubated for 1 hour in alkaline phosphatase-conjugated goat anti-mouseIgG (Kirkegaard and Perry, Gaithersburg, Md.). The blot was washed 3times in PBS-T and finally in Tris-glycine-saline, (TGS), pH 8.0.Antigenic bands were visualized by incubating the blot in TGS containing2 mg/ml napthol and 1 mg/ml phenol red (Sigma, St Louis, Mo.).

Antigen Dot Blot.

Samples were applied to nitrocellulose paper using a 96-well manifoldunder vacuum. The paper was blocked and incubated overnight in blockingbuffer containing HMAF diluted 1:500. The paper was washed 3 times withPBS-T and incubated for 1 hour in blocking buffer containing goatanti-mouse immunoglobulin gamma (Kirkegaard and Perry) labeled with ¹²⁵I(Gentry M. K. et al., Am. J. Trop. Med. Hyg., 1982, 31: 548-555), usinglabeled antibody at 10⁶ cpm/ml of blocking buffer. Following incubationwith the labeled antibody, the paper was washed 3 times with PBS-T, cutinto sample squares and counted in a clinical gamma counter(Pharmacia-LKB, Piscataway, N.J.).

Antibody Affinity Assays.

A particle fluorescence assay (PFCIA) was developed based on previousmethodologies (Scatchard, G. Ann. N.Y. Acad. Sci., 1989, 51:660-672;Schots et al. Virology, 1988, 162: 167-180) to quantitate fluorescencein an antibody-antigen binding assay using FITC-labeled purified mAbs.The amount of fluorescence, via antibody, bound to antigen adsorbed topolystyrene beads was assayed using polycarbonate IDEXX assay plates(IDEXX, Westbrook, Me.) and a PFCIA analyzer (IDEXX). Binding affinitiesof the three mAbs were measured under neutral (pH 7.0) and acidic (pH5.0) buffering conditions. Antigens tested in the assay were: rEgpderived from the cell lysate described above, partially purified rEgpobtained by column fractionation (see below) of the cell lysates, orDEN-2 virus (NGC strain). Antigen and serially-diluted FlTC-conjugatedEgp-specific mAbs (100 μg/ml) were seperately adsorbed onto polystyrenebeads (IDEXX) for 1 hour at room temperature. Protein-bound beads werewashed twice in PBS and resuspended in PBS containing 0.1% bovine serumalbumin (BSA) and 0.1% sodium azide at a final particle concentration of0.25% w/v. The assay was conducted in triplicate for each mAb dilution.For the assay, blocking buffer (PBS containing 1% BSA) was distributedinto wells of IDEXX plates followed by the addition of antigen-coatedbeads. Serial dilutions of FITC-labeled mAbs were then added to thewells and plates were incubated in the analyzer for washing (PBS, pH7.2, 0.1% BSA and 0.02% Tween) and fluorescence quantitation. Resultswere analyzed by the Ligand software program, P J Munson, Division ofComputer Research and Technology, The National Institutes of Health,Bethesda, Md.

Gel Filtration Chromatography.

Clarified supernatants of lysed, infected High five cells were strainedthrough a 0.4 micron filter and fractionated by gravity flow using acolumn of Sephadex G-100 (1.5×30 cm) or by Fast Pressure LiquidChromatography (Pharmacia) using columns of Sepharose-6 and Sepharose 12(2.5×60 cm). Fractions were collected and aliquots of the fractions wereassayed for antigenic activity by antigen dot blot assay.

Purification of rEgp by Ultracentrifugation.

Infected High five or Sf-21 cells were harvested, pelleted by low-speedcentrifugation and washed several times with PBS. The pellet wasdisrupted by sonication and clarified by low-speed centrifugation. Thesupernatant was centrifuged at 100,000×g for 90 minutes, and themicrosomal pellet was collected. The pellet was sonicated andcentrifuged at 100,000×g for 3 hours through either a step gradient of 5to 30% sucrose in PBS, or through a 30% sucrose cushion. Fractionscollected were dialyzed against PBS before testing.

Mouse Immunizations and Challenge.

Groups of ten, 4-6-week old female BALB/c mice (Jackson Laboratories,Bar Harbor, Me.) were immunized subcutaneously with doses of 0.4, 1.0and 4.0 μg of purified rEgp in 0.5ml without adjuvant or with antigenadsorbed onto Alhydrogel (Alum, Superfos Biosector, Denmark). A controlgroup of 10 mice was immunized with either PBS or 10⁴ plaque formingunits (pfu) of DEN-2 virus (NGC strain). After 28 days, animals wereboosted once with antigen, PBS or virus. Two weeks following the boost,half of the mice of each group were bled and individual sera were testedin plaque reduction neutralization assays. The other half of the mice ofeach group were challenged intracerebrally with 10⁴ pfu of DEN-2 virus(NGC strain). After 5 days, mice were sacrificed, brains wereaseptically removed, homogenized and used in a plaque assay toquantitate viral growth.

Plaque Reduction Neutralization Test (PRNT) and Viral Plaque Assay.

Mice were immunized on days 0 and 30 and bled 2 weeks following theboost. Sera collected from immunized mice at days were serially dilutedten-fold and incubated at 37° for 1 hour with 250 pfu/ml of DEN-2 virus(NGC strain). Following incubation, 2 ml aliquots of the sera-virusmixture was distributed onto duplicate monolayers of Vero cells in6-well plates. After plates were rocked for 1 hour at 37° C., monolayersan overlay of 1% melted agarose in 2× EMEM was added onto eachmonolayer. After 6 days of incubation at 37° C., a second overlay ofagarose containing a neutral red stain was applied, and plates wereincubated overnight at 37° C. Viral plaques were counted the followingday.

To quantitate viral growth, brain tissue homogenates serially dilutedten-fold were distributed onto Vero cell monolayers and incubated asdescribed above. Agarose overlays were added and viral plaques werecounted as described above.

RESULTS

Construction of Recombinant pBlueBacIII Transfer Vector.

The DEN-2 Egp gene fragment that was inserted into pBlueBacIII shown inFIG. 1. The fragment encodes the full Egp (495 amino acids) and 36 aminoacids of the C terminus of the adjacent upstream M/preM protein. Thissegment serves as a signal for membrane translocation of the Egp(Markoff, L. J. Virol. 1989, 63:3345-3352)). Synthetic primers used toamplify the gene fragment each contained 18 nucleotides complementary tospecific sequences in the DEN-2 E gene. The forward primer contains aBgl II enzyme restriction site and an ATG start codon (SEQ ID NO: 3).The reverse primer contains a Pst I enzyme restriction site and a stopcodon. The E gene fragment was cut with Bgl II and Pst I enzymes andinserted unidirectionally into the Bgl II-Pst I cloning site of thepBlueBac III plasmid placing the recombinant gene was under the controlof the AcNPV polyhedrin promoter.

Antigenicity of Baculovirus-Vectored rEgp.

To perform an epitope analysis of the rEgp, the protein suspensioncontaining rEgp and purified DEN-2 virus were reacted in an antigen dotblot assay with a panel of mAbs. The panel contained mAbs that bindeither linear or discontinuous antigenic sites, and recognize bothneutralizing and non-neutralizing epitopes. Results of the assay showedthat the rEgp reacted to every mAb in the panel (Table 1). Sincereactivities by this assay were quantitatively different for individualepitopes, binding affinities of the individual mAbs to the rEgp andnative Egp were determined. The mAbs selected for affinity assays, 2H3,4G2, and 9D12, demonstrated weak (2H3) to strong (9D12) binding to therEgp in the antigen dot blot assay. Table 2 shows that the bindingaffinities of individual mAbs for rEgp and partially purified rEgp wascomparable to their affinities for virus. Binding assays conducted atboth neutral and slightly acidic pH demonstrated that these epitopeswere not affected by pH.

TABLE 1 Antibody binding of the dengue-2 recombinant envelope proteinexpressed by baculovirus. Reactivity with antigen^(b) Antibody^(a)ACNPV-E ACNPV-prME DEN-2 Virus ACNPV 3H5^(d) 13.6^(c) 10.1 7.5 1.49D12^(d,e) 12.3 12.1 9.0 1.0 13B7 10.5 4.1 5.6 3.6 4E5^(d) 8.6 6.6 10.51.0 2H3^(d) 4.9 2.5 11.5 1.9 4G2^(d,e) 8.5 5.0 16.8 1.0 1B7^(d,e) 5.13.1 8.9 1.2 HMAF 13.5 17.6 7.0 1.2 HCS 12.5 NT 12.5 1.6 ^(a)Antibodieswere diluted 1:100 (mAbs) or 1:500 (anti-DEN-2 hyperimmune mouse ascitesfluid, HMAF; or convalescent human sera, HCS). ^(b)Antigenicityreactivity of extracts from High-5 cells infected with recombinantbaculovirus clones containing DEN-2E (ACNPV-E) or prME (ACNPV-prME)genes, tested by antigen dot blot assay. Purified DEN-2 virus served asthe positive control in the assay. Protein extracted from High-5 cellsinfected with wild-type baculovirus served as the negative control.^(c)Antigen-antibody biding was detected by ¹²⁵I-labeled goat anti-mouseimmunoglobulin. Data for each mAb and HMAF represents an average ofthree separate experiments; and for HCS, one experiment. Results aregive as cpm × 10³. ^(d)Antibodies which neutralize virus infectivity invitro (Henchal et al. Am J. Trop. Med. Hyg. 1985, 34:162-167).^(e)Antibodies which recognize conformational epitopes (Henchal et al.Am J. Trop. Med. Hyg. 1985, 34:162-167; Megret et al. Virology, 1992,187:480-491).

TABLE 2 Binding affinity of monoclonal antibodies to recombinant andnative dengue-2 envelope proteins. Affinity binding of mAbs 9D12, 2H3,and 4G2 at pH 5.0: Antigen^(a) 9D12 2H3 4G2 Purified 0.4 × 10⁻⁶ 3.2 ×10⁻⁶ 2.3 × 10⁻⁶ Lysate 0.5 × 10⁻⁶ 1.0 × 10⁻⁶ 2.9 × 10⁻⁶ Virus 5.2 × 10⁻⁶2.0 × 10⁻⁶ 1.3 × 10⁻⁶ ^(a)Antigens were either partially-purifiedrecombinant E protein, lysates of cells infected with the E-proteinrecombinant baculovirus, or purified DEN-2 virus.

Anaylsis of the antigenic properties of the full DEN-2 rEgp expressed inthis study by baculovirus demonstrated that properly conformed proteinscan be produced in this system. This was evidenced by the strongreactivity of the rEgp with mAbs that represented both linear andconformational-dependent epitopes within the native protein. Bindingaffinities of selected mAbs to native epitopes were not modified in therecombinant protein.

The mAb binding assays qualitatively demonstrate that native proteinepitopes were preserved on the recombinant E protein.

Gel Filtration Analysis of DEN-2 rEgp Particles.

The DEN-2 Egp was expressed from baculovirus in High-five and Sf-21cells. Cells were lysed by sonication in PBS containing 0.1% sarkosyl.Gel filtration of the cell lysates shows that the majority of rEgpproduced by baculovirus had self-aggregated to form high molecularweight particles. Protein separation profiles for infected cell lysatesare shown in FIGS. 2, 3, 4 and 5. Antigenic reactivity with anti-DEN-2HMAF is distributed among nearly all fractions passed through G-100Sephadex, with a major antigenic peak eluting at the position ofcalibration standard thyroglobulin, molecular weight (mol wt) 670kilodaltons (kd). Similar results were obtained for gel FPLC usingSuperose 6 (FIG. 3) and Superose 12 (FIGS. 4 and 5). The rEgp was elutedin the void volume of the Superose 6 column (molecular weight exclusion,5×10⁶ kd) in fractions 8 through 11 compared to the calibration standardthyroglobulin which was eluted in fractions 13 and 14. Similarly, therEgp eluted in the void volume of the Superose 12 column (mol wtexclusion, 3×10⁵).

The role of sarkosyl and sonication in disruption of rEgp particles wasalso examined during Superose 12 chromatography. By equilibrating columnin increasing amounts of sarkosyl (0.1 to 3.0%), protein elutionprofiles were shifted, however position major antigenic peak associatedwith rEgp was not altered by sarkosyl (FIGS. 4A, B, C, D). Sonicationfor up to 30 minutes did, however, partially disrupt rEgp aggregates aswell as other high molecular weight protein aggregates (FIGS. 5A, B, andC).

Purification of E Particles.

Gel filtration results indicated that rEgp aggregates could be separatedfrom the majority of cellular proteins based on their large size.However, yield of partially purified rEgp produced in this manner wererelatively low and the process was slowed by frequent necessity to cleanthe column matrix. Aggregated rEgp particles were therefore purifiedfrom other cellular components by differential centrifugation using asucrose cushion. In initial experiments, the microsomal fraction ofinfected cell lysates was collected by ultracentrifugation, sonicated,and centrifuged through a 5-30% sucrose step gradient Fractionscontaining 500 μl were concentrated, dialyzed and analyzed forreactivity with anti DEN-2 mouse HMAF. FIG. 6 shows that very highantigenic activity was present in the gradient pellet, compared torelatively small amount of antigenic activity that was distributed intoseveral gradient fractions. Since the majority of E antigen was presentin the 5-30% sucrose gradient pellet, E protein aggregates were purifiedby centrifugation of the microsomal fraction through a 30% sucrosecushion.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting.

Proteins that were pelleted through the 30% sucrose cushion wereanalyzed by SDS-PAGE and western blotting. As shown in FIG. 7A, thispellet contained three protein bands that stained with Coomassie blue ona 10% reduced SDS-polyacrylamide gel. A western blot of a non-reduced10% gel loaded with identical samples revealed three antigenic bandsthat appear to correspond to the three protein-stained bands (FIG. 7B).These bands seen on the protein gel and on the blot migrate closetogether, and are likely to represent varying degrees of glycosylationof the rEgp.

Immunogenicity of the Purified rEgp.

The purified rEgp particles were tested in immunogenicity trials inmice. Previously it was shown that a cellular lysate containingbaculovirus vectored rEgp was fully reactive with native E-specificmonoclonal antibodies and induced a low titer of neutralizing antibodyin mice. Table 3 shows results from immunization of mice with purifiedrEgp. Mice responded to immunization by production of neutralizingantibodies. Table 3 shows that a non-adjuvanted immunizing dose of 4 μginduced production of neutralization antibodies. This response wasboosted several fold when rEgp was pre-adsorbed to Alum, and wasequivalent to titers induced by live virus. The pre-absorption to alumalso increased the response with 1 μg to a detectable level (Table 3).

TABLE 3 PRNT₅₀ in mice immunized with baculovirus expressed DEN-2 Eprotein. rEgp with Alum rEgp/no adjuvant Dose 4 μg Dose 1 μg Dose 4 μgDose 1 μg DEN-2 NGC Control >850 458 472 <13 633 <13 >850 280 233 <13526 <13 441 473 538 <13 480 >850 476 261 <13 622 540 788  23 ND 574*Vaccination schedule: Day 1 and day 30. Bled two weeks after the seconddose.

Mice immunized with purified non-adjuvanted and adjuvanted rEgp weretested in a challenge assay with live virus. Table 4 shows results forgrowth of DEN-2 challenge virus in immunized and control mice.

TABLE 4 Percent protection measured by reduction of dengue virus in thebrains of immunized. intracerebrally challenged mice Immunization¹Percent reduction²   4 μg with alum 88.5 ± 35.1   1 μg with alum  97 ±4.2 0.25 μg with alum 92.6 ± 5.1    4 μg without alum 83.4 ± 40.2   1 μgwithout alum 78.5 ± 24.7 0.25 μg without alum 96.8 ± 3.8  live virus 100± 0  none  0 ± 86 ¹Mice were immunized at days 0 and 30 with indicatedamounts of recombinant dengue 2 envelope protein or with live dengue 2virus. Control mice were not immunized. ²Mice were inoculatedintracerebrally with 10,000 pfu of mouse adapted dengue 2 virus twoweeks after the last immunization. Five days later, mice were euthanizedand the brains removed for quantitation of dengue virus in the brain.The percent reduction was calculated by multiplying 100 times theformula (control-plagues/control) where control is the mean of the virusplaques in un immunized mice and plaques is the plaques measured inindividual mice. Results are displayed as the mean ± the standarddeviation for the mice in each group.

Table 4 shows that the mean number of viral plaques obtained from brainsof all groups of immunized mice were greatly reduced compared to thatobtained in unimmunized mice. Mean number of plaques obtained for miceimmunized with adjuvanted antigen (groups 1, 2 and 3 mice, immunizedwith 4, 1, and 0.4 μg of rEgp, respectively), were significantly lowerthan those obtained for mice immunized with non-adjuvanted rEgp.

1. A composition comprising a DNA that encodes a protein that induces animmune response in mammals against Dengue-2, wherein said DNA comprises,SEQ ID NO: 3, SEQ ID NO: 1 and SEQ ID NO:
 4. 2. A composition comprisingan RNA molecule that encodes a transcript of SEQ ID NO: 3, SEQ ID NO: 1and SEQ ID NO: 4, wherein the encoded protein produced there fromelicits an immune response in mammals against Dengue-2.
 3. Thecomposition of claim 1, further comprising at least one other DNAfragment comprising genes from at least one other serotype of denguevirus.
 4. The composition according to claim 2, further comprising atleast one other DNA or RNA fragment comprising genes from at least oneother serotype of dengue virus.
 5. The composition of claim 1, furthercomprising a pharmaceutically acceptable excipient.
 6. The compositionof claim 2, further comprising a pharmaceutically acceptable excipient.7. The composition of claim 1, further comprising at least one other DNAfragment from at least one other serotype of dengue virus.
 8. Thecomposition of claim 2, further comprising at least one other RNAfragment from at least one other serotype of dengue virus.
 9. Thecomposition of claim 1, wherein said DNA contains one or morerestriction sites.